This invention is concerned with novel covalent peptide-protein conjugates and a method of making and a method of using such conjugates. Principal Neutralizing Determinant (PND) peptides, having the property of binding to antibodies capable of neutralizing Human Immunodeficiency Virus (HIV), are conjugated to a carrier which is comprised of an immunogenic protein or protein complex, preferably the Outer Membrane Protein Complex (OMPC) of Neisseria.
The term "neutralizing" as applied to antibodies means that viral exposure to such antibodies, whether in vitro or in vivo, results in the attenuation or abrogation of any or all of the recognized virus-mediated pathophysiologic functions characteristic of HIV infection and disease including cellular fusion, cellular infection, CD4 receptor bearing cell depletion, and viral proliferation. Neutralizing antibodies meeting these criteria have been detected in the sera of HIV-infected patients and have been induced in animals and humans by immunization with diverse HIV related antigens.
Attenuated or killed whole HIV, HIV subunit proteins, live recombinant vaccinia virus containing incorporated HIV genetic material, and HIV specific peptides have all been evaluated for potential use as protective or therapeutic post-infection immunogens. The whole-virus approach is attended by the danger, however small, of producing an active infection in vaccine recipients, while the subunit-protein-vaccine approach has mer with limited success in the induction of virus-neutralizing antibodies. The live-recombinant-vaccinia-virus vaccine approach has promise, but the dangers inherent in the introduction of a live virus, however benign, especially into an already immunocompromised recipient, are obvious. Thus far, peptide based immunogens hold the most promise. The following references provide a general overview of ongoing vaccine evaluations: Lasky, L. A., Crit. Rev. in Immunol. 9, 153 (1989); Garrison, L., Clements, L. M., Comprehensive Therapy 15, 47 (1989); Dalgleish, A., Drugs of Today, 25, 709 (1989); Schulhafer, E. P., Verma, R. S., In Vivo 3, 61 (1989); Fauci, A. S., et al., Annals of Internal Medicine 110, 373 (1989); Rosenberg, Z. F., Fauci, A. S., Advances in Immunol. 47, 377 (1989); and Snart, R. S. AIDS 2, S107 (1988).
Peptides of interest in this invention, hereinafter referred to as Principal Neutralizing Determinants (PNDs), have been identified which are capable of eliciting HIV-neutralizing immune responses in mammals. Although immunogenicity can be conferred on other HIV related or unrelated peptides upon conjugation according to this invention, of particular interest here is the PND located in the HIV IIIB and in most other HIV isolates, such as the MN isolate, at or near the amino acids between 296 and 341 of the HIV envelope glycoprotein, gp120 [numbering according to the scheme of Ratner et al., Nature 313, 277 (1985)]. Although the amino acid sequence in this region is variable across HIV isolates, the inter-isolate conserved core amino acid sequence, Gly-Pro-Gly (GPG), appears in over 90% of the isolates tested in one study, while the sequence Gly-Pro-Gly-Arg-Ala-Phe (GPGRAF) appears in a large number of common isolates [Goudsmit, J., Aids 2, S41 (1988)]. Less highly conserved amino acids appear on either side of the GPG trimer. A minimum of between 5 and 8 amino acids, including the GPG, appears to be necessary to induce an HIV neutralizing response [Javaherian et al., PNAS U.S.A. 86, 6768 (1989); Goudsmit et al., Res. Virol. 140, 419 (1989)].
Linear or cyclic peptides may be utilized to make conjugates which generate HIV neutralizing immune responses. Small divergences in amino acid sequence, for example the substitution of a valine for an alanine, or an aspartate for a glutamate, may in some cases give rise to peptides capable of eliciting similar immune responses. Furthermore, peptides having conserved tertiary structures but having divergent primary structures, as in a series of cyclic PND peptides (cPNDs), may give rise to similar immune responses.
Because HIV is known to be transmitted in either a cell-free or a cell-associated form, it may be an essential requirement that peptidyl epitopes be capable of priming both B-cell- and T-cell- mediated immune responses, such as antibody production and antibody-dependent cellular cytotoxicity, in order to be useful as anti-HIV immunogens. Peptide sequences from the gp120 region described above have been shown to be capable of inducing both types of immune responses [Goudsmit, J., AIDS 2, S41 (1988)].
In addition, in order to generate a useful anti-HIV vaccine, PND peptides, which are generally poorly immunogenic on their own, often must be conjugated to a carrier in a reproducible and quantifiable fashion. Unconjugated peptides are not only poor inducers of B-cell-mediated antibody production, they are also weak inducers of protective T-cell responses. The instant invention overcomes these problems by providing novel immunological conjugates of the PND peptides and immune enhancers.
In U.S. Pat. No. 4,695,624, Marburg et al. disclosed conjugation chemistry for covalently coupling the Haemophilus influenzae b capsular polysaccharide, polyribosyl ribitol phosphate (PRP) to the OMPC of Neisseria meningitidis. Such conjugates were capable of eliciting anti-PRP immune responses and were useful as immunogens to prevent Haemophilus influenzae b infections. The conjugates of this invention raise a completely novel immune response against the HIV PND peptides which are absent in the conjugates of the U.S. Pat. No. 4,695,624.
The novel conjugates of this invention are useful for inducing mammalian immune responses against the peptidyl portion of the conjugate. Where the peptide component of the coconjugate represents an HIV PND peptide, or a peptide capable of eliciting immune responses which recognize HIV PND peptides, the conjugates are useful for inducing anti-HIV PND peptide antibodies in mammals, for inducing HIV-neutralizing antibodies in mammals, or for vaccinating humans prior to or after contraction of HIV infection or disease including AIDS.